The papillomaviruses cause benign tumors of epithelial cells in both humans and animals, and some cases these tumors progress to malignancies. The viruses are poorly understood because they do not productively infect cells in tissue culture, but mouse cells in culture can be transformed to tumorigenicity by bovine papillomavirus, type I (BPV) or by BPV DNA. The long term goal of this project is to use genetic approaches to analyze cell transformation by BPV. A number of experimental approaches will be used. First, the human DNA sequences that stimulate BPV tranformation will be precisely localized and their mechanism of action will be studied. Second, viral mutants defective for transformation will be isolated and characterized. Because some BPV recombinants replicate as plasmids in mouse cells and bacteria, it may be possible to identify conditionally-defective viral mutants in transformed mouse cells and to recover them in bacteria for detailed analysis. In addition, nucleotide substitutions will be constructed at predetermined locations in the viral overlapping translational reading frames to attempt to resolve their functions. Cells transformed by various BPV plasmids will be tested to determine if any express a subset of the properties found in fully-transformed cells. DNA immunoprecipitation and gradient-of-label experiments will be performed to localize the origin of viral DNA replication in transformed mouse cells. Finally, BPV vectors will be used to search for homologous recombination between a plasmid-borne segment of the mouse HPRT gene and its chromosomal counterpart. If successful, these experiments may help indicate how the acquisition of a few genes can convert a normal cell into a maligmant one. Increased understanding of the biology of BPV will also facilitate its use as a eucaryotic vector.